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Ultrasensitive Immunoassay of Proteins Based on Gold Label/Silver Staining, Galvanic Replacement Reaction Enlargement, and in Situ Microliter-Droplet Anodic Stripping Voltammetry Xiaoli Qin,† Ling Liu,† Aigui Xu,† Linchun Wang,‡ Yueming Tan,† Chao Chen,† and Qingji Xie*,† †

Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (MOE of China), National & Local Joint Engineering Laboratory for New Petro-Chemical Materials and Fine Utilization of Resources, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China ‡ Liuzhou Traditional Chinese Medicine Hospital, Liuzhou 545001, China S Supporting Information *

ABSTRACT: An ultrasensitive metal-labeled amperometric immunoassay (MLAI) method of proteins is reported, on the basis of multiple cycles of gold label/silver staining and galvanic replacement reactions (GRRs), followed by simultaneous chemical dissolution/cathodic preconcentration of silver for in situ microliter-droplet anodic stripping voltammetry (ASV) detection on the immunoelectrode. Briefly, antibody 1 (Ab1), bovine serum albumin (BSA), antigen, and Au nanoparticles functionalized with antibody 2 (Ab2−AuNPs) were successively anchored on a Au-plated glassy carbon electrode (GCE) to form a sandwich-type immunoelectrode (Ab2−AuNPs/antigen/BSA/Ab1/Auplate/GCE). Silver was selectively stained on the catalytic AuNPs surfaces through chemical reduction of silver cations by hydroquinone (gold label/silver staining). A beforehand “potential control” in air and then injection of 5 μL of 25% aqueous HNO3 on the immunoelectrode surface for dissolution of the stained silver enabled rapid cathodic preconcentration of atomic silver onto the electrode surface as entirely as possible from the lysate, followed by ASV detection of silver. Multiple GRRs between HAuCl4 and the stained silver were used to amplify the signal, and the stoichiometry of this GRR at the electrode surface was clarified by an electrochemical quartz crystal microbalance. Under optimized conditions, this method was used for ultrasensitive quantitative analysis of human immunoglobulin G (IgG) and human α-fetoprotein (AFP), giving limits of detection (LODs, S/N = 3) of 0.2 fg mL−1 for IgG and of 0.1 fg mL−1 for AFP (equivalent to five molecules in the 6 μL samples employed for analysis of both proteins), ultrahigh sensitivity, excellent selectivity, and little consumption of reagent. The thermodynamic feasibility of such a single-molecule-level amperometric immunoassay (interface-based bioassay) is theoretically proven on the basis of a deduction of the thermodynamic equilibrium of the immunological reactions occurring at the electrode|solution interface.

1. INTRODUCTION Innovation of ultrasensitive bioanalysis methods has received great attention in many areas, including clinical assay, foodsafety analysis, and environmental analysis. Molecular and nanomaterial biolabeling techniques for signal output and amplification have been widely used in bioanalysis.1−4 The nanomaterials of metals and their compounds have been often used as biolabels for bioelectroanalysis, because the metal components can be sensitively and conveniently determined by anodic stripping voltammetry (ASV). The metal-labeled amperometric immunoassay (MLAI) involving a sandwichtype immunointerface has been reported to be a promising method for bioanalysis,5−8 but the relevant ASV analysis was usually conducted on the basis of a solution-replacement protocol, by which the detection sensitivity is limited due to the solution-dilution effect.9 Virtually, the metal-nanomaterialmodified electrode also acts as a good experimental platform to advance nanoscience and nanotechnology.10−13 Hence, © XXXX American Chemical Society

enhancing the amperometric signals of the electrode-supported metal nanomaterials is obviously an interesting and important topic for characterization and applications of metal nanomaterials. From the microscopic point of view, the movement of extranuclear electrons constitutes one of the core scientific issues in chemistry and its downstream disciplines, such as molecules/atoms-based biology and material sciences.9 In our opinion, the electron-movement behaviors in chemistry can be divided at least into four classes, each responsible for some important chemistry branches: (1) energy-level transitions of valence and inner-shell electrons for various atomic and molecular spectroscopy techniques in chemistry; (2) weak electronic interactions for any known weak inter/intraReceived: December 8, 2015 Revised: January 15, 2016

A

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for biolabeling in bioanalysis.23−26 In addition, the gold-label/ silver-staining technique has been widely used for signal amplification of bioaffinity-based bioanalysis, since silver can be stained solely on the surface of catalytic Au nanoparticles (AuNPs) through chemical reduction of silver cations by hydroquinone (HQ).6,27,28 The GRR between a Au salt and the stained silver may enhance the size of the immunolabeled AuNPs, and repeated GRRs can increase the quantity of catalytically stained silver and thus amplify the MLAI signal based on the ASV of silver. However, no such attempts have been reported for ultrasensitive bioelectroanalysis so far. Herein, we report a sandwich-type MLAI method with signal amplification for ultrasensitive detection of proteins (Scheme 1), on the basis of gold label/silver staining, GRRs, and

molecular interactions and the interactions-based chromatographic separation/analysis; (3) strong electronic interactions (electron sharing) for covalent-bond formation and synthetic chemistry; and (4) complete electron gains and losses for electrochemistry, redox chemistry, ion chemistry, and mass spectrometry. Hence, in this sense, we may regard chemistry as the science mainly dealing with and making use of extranuclear electrons. The electrode in electrochemistry is an excellent platform to study gains and losses of valence electrons, which can directly return important information on the electron transfer of the atoms and/or molecules at the electrode| electrolyte solution interface. Electrochemistry has played an irreplaceably important role in revealing the fact that the efficient distance for valence electron transfer/communication in the ion-conducting but electron-insulating electrolyte phases is on the nanometer scale, as discussed in detail in Scheme S1 of the Supporting Information (SI). The electrochemistryrevealed nanometer-scale distance required for an efficient valence electron transfer reaction may be universally interesting and important in deepening our understanding of the maximum distance between reactant atoms and/or molecules required for occurrence of the conventional chemical reactions or weak intermolecular/interatomic interactions, e.g., a nanometer-scale distance for reactant particle “collision” may be sufficiently short for occurrence of a chemical reaction. Obviously, driving the electroactive species to move close to or even touch the electrode surface is very important in conducting an electrochemical reaction and fulfilling an analytical task occurring on the electrode with enhanced sensitivity, as reported for some electrochemical biosensors based on target-responsive structural switching and folding of functional nucleic acids.14,15 Accordingly, in the case of metal biolabels for bioelectroanalysis, we can realize an enhanced signaling in an amperometric sandwich bioassay simply by simultaneous chemical dissolution/cathodic preconcentration of the metal biolabels for in situ ASV analysis on the bioelectrode, and this protocol can efficiently avoid the spatial hindrance of the electron-insulating sandwich biostructure on the bioelectrode surface against redox electron transfer, as demonstrated in Scheme S1 (SI). Development of ultrasensitive analytical approaches to single-molecule-level detection (SMLD) represents an important research direction and a possible ultimate goal in quantitative analytical chemistry.16−18 To date, many efforts have been devoted to developing SMLD methods, mainly involving advanced optical and electrochemical analysis ones (Table S1, SI). Obviously, boosting the analytical signals by innovation and optimization of the detection principles, methodology, and materials is very important in SMLD research. In our opinion, the MLAI involving a sandwich-type immunointerface should have the potential for SMLD of proteins, since a deduction based on the thermodynamic equilibrium of the immunological reactions occurring at the electrode|solution interface can prove the thermodynamic feasibility of such an SMLD, as discussed in detail later. The galvanic replacement reactions (GRRs) between lessnoble metals (reductants) and the salts of more-noble metals (oxidants) have been widely employed in areas ranging from materials to environment and energy. For instance, the GRR technique has been used to synthesize various noble metal (e.g., Au, Pt, and Pd) nanomaterials with controllable structures and compositions for enhanced applications,19−22 including the solution-phase preparation of specially shaped nanomaterials

Scheme 1. Illustration of Immunoelectrode Preparation (a) and Key Electrochemical Steps (b) of our MLAI Method

enhanced cathodic preconcentration/in situ ASV analysis of the metal nanomaterial biolabels. The motivation is based on the fact that after immunologically anchoring the second antigen (Ab2) labeled with metal nanoparticles to form a sandwich-type immunointerface on the electrode, the metal label after selective and efficient size enhancement should output a sufficiently large ASV signal for ultrasensitive bioanalysis of antigen proteins. Our MLAI method can show limits of detection (LODs, S/N = 3) of 0.2 fg mL−1 for human immunoglobulin G (IgG) and 0.1 fg mL−1 for human αfetoprotein (AFP) (i.e., five molecules in the 6 μL samples taken for both proteins) under optimized conditions, which is a large improvement versus the methods reported for the two proteins before (Table S2, SI). In addition, the theoretical feasibility of such a single-molecule-level amperometric immunoassay is discussed on the basis of the thermodynamic equilibrium of the immunological reactions occurring at the sandwich-type immunoelectrode interface. The basic procedures of our MLAI method are depicted in Scheme 1, and a synoptic introduction is given as follows. Briefly, the first antibody (Ab1) was adsorbed on an Auelectroplated glassy carbon electrode (GCE), bovine serum albumin (BSA) was used to block the nonspecific binding sites, and then the target antigen was immobilized by the B

DOI: 10.1021/acs.jpcc.5b12004 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C immunological reaction. The Ab2 labeled with AuNPs (Ab2− AuNPs) was immunogically captured onto the electrode to form a sandwich-type immunointerface. Afterward, silver was stained selectively on the catalytic AuNPs surfaces through chemical reduction of silver cations by hydroquinone (HQ).29,30 The GRR between the stained silver on the immunoelectrode and aqueous HAuCl4 was allowed to occur when needed, yielding size-enlarged Au and AgCl structures. The silver staining−GRR operation could be repeated several cycles when needed. As highlighted in Scheme 1b, a cathodic potential (−0.3 V here) was first applied on the immunoelectrode in air (no addition of an electrolyte solution to electrically connect the electrodes involved), and 5 μL of 25% aqueous HNO3 was then added to connect the glassy carbon working electrode (WE), the KCl-saturated calomel reference electrode (RE), and the platinum wire counter electrode (CE); to destroy the electrode-supported biostructure (Scheme S2, SI); and to dissolve the stained silver for diffusion-controlled cathodic preconcentration of atomic silver onto the immunoelectrode substrate. Finally, 45 μL of 0.6 M aqueous KNO3 was added for differential pulse ASV analysis. As shown in Scheme S2 (SI), the beforehand cathodic “potential control” in air and the use of a small volume of acid can minimize the diffusion-layer thickness to rapidly electrodeposit the signaling silver onto the immunoelectrode surface as entirely as possible from the lysate containing Ag+ ions and collapsed AgCl nanostructures, which can greatly enhance the subsequent ASV signal for bioanalysis. Note that the potential control in air is safe for an electrochemical instrument working in its potentiostatic mode but may damage the instrument running in its galvanostatic mode. The potential control in air is not so commonly used in analytical chemistry and electrochemistry, but this special procedure was used here to effectively amplify the bioelectroanalysis signal.

for convenience, respectively, as reported before.32−35 Bovine serum albumin (BSA) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). AgNO3, HQ, and trisodium citrate were purchased from Shanghai Chemicals Station (Shanghai, China). The washing and blocking buffer solution for immunoassay was 0.01 M phosphate buffer saline (PBS, pH 7.4, 10 mM NaH2PO4−Na2HPO4 + 0.15 M NaCl). Citrate buffer was an aqueous mixture of 0.243 M C6H8O7·H2O and 0.163 M Na3C6H5O7·2H2O (pH 3.5). The silver-staining solution prepared daily was composed of 1.0 g of HQ, 35 mg of AgNO3, 50 mL of ultrapure water, and 50 mL of the above citrate buffer. All other chemicals were of analytical grade or better quality. Milli-Q ultrapure water (Millipore, ≥18 MΩ cm) was used throughout. The clinical serum samples were donated by the Liuzhou Traditional Chinese Medicine Hospital, Guangxi Zhuang Autonomous Region, China, and the AFP level in each sample was analyzed by chemiluminescence in the hospital. 2.2. Procedures. The sliver nanoparticles were prepared as reported.36 All glassware was soaked in aqua regia (VHNO3:VHCl = 1:3), rinsed with ultrapure water, and then oven-dried prior to use. In a 50 mL Erlenmeyer flask, 8.5 mg of AgNO3 was dissolved into 50 mL of H2O and brought to boiling. Then, 1 mL of 1% (w/v) trisodium citrate solution was quickly added into the boiling solution and the solution was kept boiling for ca. 1 h. The prepared silver sols were greenish yellow and had an absorption maximum at 420 nm,36 as shown in Figure S1 (SI). Considering the loading of antibody and the steric hindrance effect in the immunoreactions, we prepared the AuNPs of 13 ± 4 nm diameter for biolabeling according to the literature.37 The glassware was cleaned as above. Briefly, 100 mL of 0.01% (w/v) aqueous HAuCl4 was brought to boiling under vigorous stirring, and 2.5 mL of 1% (w/v) aqueous trisodium citrate was quickly added to the boiling solution. When the solution turned deep red in color, indicating the formation of AuNPs, boiling was pursued for an additional 10 min. The heating source was removed, and the solution was left stirring and cooled down to room temperature. Then, 10 μg of Ab2 was added to 1.0 mL of this AuNPs dispersion and gently mixed at 4 °C overnight. After centrifugation at 4800 rpm for 30 min, the supernatant was discarded and the sediment was washed with 0.01 M PBS (pH 7.4). After another centrifugation and discarding of the supernatant, the resulting Ab2−AuNPs were redispersed into 0.5 mL of 0.01 M pH 7.4 PBS containing 1.0% (w/v) BSA to block the nonspecific sites and stored at 4 °C prior to use. The GCE was carefully polished with aqueous alumina slurries (Al2O3) by stepwise decreasing the particle size (0.5 and 0.05 μm). After being thoroughly rinsed with water, the polished electrode was ultrasonically treated sequentially in water, ethanol, and water, each for 5 min, to remove residual alumina powder. Then, the GCE was treated with concentrated sulfuric acid for 15 s. Afterward, the GCE was subjected to electrochemical rinsing in 0.50 M aqueous H2SO4, i.e., the GCE was scanned between −1.0 and 1.0 V vs SCE at 100 mV s−1 for a sufficient number of cycles to obtain reproducible cyclic voltammograms. As shown in Scheme 1, first, Au was electroplated on the cleaned GCE (Auplate/GCE) by double potential step pulse electrolysis from 1.1 to 0 V with a pulse width of 0.25 s in 0.50 M aqueous H2SO4 containing 2.0 mM HAuCl4,32 and the total

2. EXPERIMENTAL SECTION 2.1. Instrumentation and Chemicals. All electrochemical experiments were conducted on a CHI660C electrochemical workstation, and a three-electrode electrolytic cell was used. A disk GCE with 3.0 mm diameter and a platinum wire of 0.1 mm diameter (CH Instruments, Inc.) served as the WE and the CE, respectively. The RE was a KCl-saturated calomel electrode (SCE), which was separated from the electrolytic solution by a Luggin capillary filled with saturated KNO3. All potentials are cited versus SCE. A computer-interfaced HP4395A impedance analyzer was employed in the quartz crystal microbalance (QCM) experiments.31 AT-cut 9 MHz piezoelectric quartz crystals (PQCs) with 12.5 mm wafer diameter (model JA5, Beijing Chenjing Electronics Co., Ltd.) were used. The Au electrode of 6.0 mm diameter (key-hole configuration, area = 0.29 ± 0.01 cm2) on one side of the PQC was exposed to the solution and served as the working electrode, while that on the other side faced air. The UV−vis spectra were recorded on a UV-2450 spectrophotometer (Shimazu Co.). Scanning electron microscopy (SEM) images and energy-dispersive X-ray (EDX) spectra were collected on a JEM-6700 field emission scanning electron microscope equipped with an EDX spectrometer. Goat anti-human IgG (anti-IgG) and IgG (MW = 150 000 Da) were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. Monoclonal mouse anti-human AFP (anti-AFP) and AFP (MW = 69 000 Da) were purchased from Beijing Key Biotech. Co., Ltd. Note that anti-IgG and anti-AFP were used here as both Ab1 and Ab2 in their immunoelectrodes C

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3. RESULTS AND DISCUSSION 3.1. Signaling Efficiency of Our MLAI Method in Simulation Experiments. To compare the signaling efficiency (δ, defined as in eq 1) of our MLAI method with those of protocols similar to our MLAI but without the beforehand potential control in air, the conventional solutionreplacement protocol and the direct anodic stripping protocol, we conducted simulation experiments simply by cast-coating an appropriate amount of AgNPs (nAgNPs‑cast in mol) on a GCE and then detecting the recovered amount of the cast-coated AgNPs (nAgNPs‑LSV in mol) by linear sweep voltammetry (LSV) and the Faraday law

time for this experiment was optimized to be 180 s. Second, PBS (6.0 μL) containing 1.0 mg mL−1 Ab1 was dropped on the Auplate/GCE and kept at 4 °C overnight to realize a saturated adsorption of Ab1 (Ab1/Auplate/GCE). Third, excess Ab1 was washed away with the washing buffer, and the Ab1/Auplate/GCE was then exposed to PBS (6.0 μL) containing 3% BSA for 1 h to block the nonspecific binding sites (BSA/Ab1/Auplate/GCE). After another washing with the buffer, the BSA/Ab1/Auplate/ GCE was stored in PBS at 4 °C, when not in use. The assay of antigen is illustrated in Scheme 1. First, the immunoelectrode was incubated in 6 μL of PBS containing antigen (a tube with a PBS drop was covered on the WE to avoid solvent evaporation) at 37 °C for 1 h to form antigen/ BSA/Ab1/Auplate/GCE. After being rinsed with the washing buffer, this electrode was incubated in 6 μL of PBS containing Ab2−AuNPs at 37 °C for 40 min to form Ab2−AuNPs/ antigen/BSA/Ab1/Auplate/GCE and then rinsed three times with PBS to remove the nonspecific binding species. After removing the rinsing solution, 6 μL of silver-staining solution was dropped on the Ab2−AuNPs/antigen/BSA/Ab1/Auplate/ GCE, which was incubated at room temperature for 30 min, yielding a silver/Ab2−AuNPs/antigen/BSA/Ab1/Auplate/GCE. The electrode was washed with ultrapure water three times. Then, 6 μL of 5.0 mM aqueous HAuCl4 was added on the immunoelectrode to allow 10 min GRR, yielding size-enlarged Au nanoparticles and AgCl [(Au−silver)1/Ab2−AuNPs/antigen/BSA/Ab1/Auplate/GCE]. Here the 6 μL volume was selected to save reagent and as the best electrode coating. The silver staining−GRR operations were repeated several cycles to maximize the silver-staining quantity [silver−(Au− silver)j/Ab2−AuNPs/antigen/BSA/Ab1/Auplate/GCE; here the subscript j denotes the number of the operation cycles]. The final immunoelectrode was obtained and stored in a dry environment prior to use. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in PBS containing 1.0 mM K4Fe(CN)6 and 1.0 mM K3Fe(CN)6 as well as QCM experiments were performed for better understanding of various electrode modification steps. For the EIS measurements, the working electrode potential was fixed at the formal potential of the Fe(CN)63/4− redox couple after being conditioned at this potential for at least 100 s. Differential pulse ASV for metal immunoassay was conducted as follows. First, we applied −0.3 V cathodic potential in air, and this potential is sufficiently negative to realize diffusion-controlled silver electrodeposition in the solution under our experimental conditions. Then, the stained silver was dissolved by addition of 5 μL of 25% aqueous HNO3, the cathodic preconcentration of atomic silver was simultaneously started and lasted for 600 s, and 45 μL of 0.6 M aqueous KNO3 was then added at the last 30 s to inhibit the interference of Cl−. Differential pulse ASV from −0.3 to 0.7 V with 4 mV potential step, 50 mV amplitude, and 50 ms pulse width was performed to record the ASV current of atomic silver. The conventional solution-replacement method was conducted as follows. The acidic lysate (5 μL) was transferred from the electrode surface into a 5 mL beaker containing 995 μL of 0.6 M KNO3 and 0.1 M HNO3 (or just 25% HNO3) as the electrolyte solution, followed by cathodic preconcentration on another Auplate/GCE prepared as above under solutionstirred condition and then ASV analysis.

δ = nAgNPs‐LSV /nAgNPs‐cast = Q p/(zFnAgNPs‐cast)

(1)

where Qp is the charge under the LSV stripping peak of silver, z is the number of electrons transferred (z = 1 here), and F is the Faraday constant (96485.3 C mol−1). The anodic stripping LSV curves and corresponding δ values as functions of the cathodic preconcentration time are shown in Figure 1. After acidic dissolution of AgNPs, 600-s cathodic

Figure 1. δ versus preconcentration time curves (A and insets of C and D, n = 3) and anodic stripping LSV curves of the AgNPs castcoated on GCE (B, C and D, 100 mV s−1) for our MLAI method (B and curve a in A), the protocol similar to our MLAI but without the beforehand potential control in air (C and curve b in A), and conventional solution-replacement protocol (D and curve c in A). Conditions: 10 μL of 1 mM AgNPs dispersion for cast-coating. See the text for other parameters.

preconcentration, and then LSV stripping in 5 μL of 25% HNO3, the δ of our MLAI method was as high as 82.1%. In contrast, the protocol similar to our MLAI but without the beforehand potential control in air for silver preconcentration only gave δ = 12.9% after 600-s preconcentration, highlighting the importance of the beforehand potential control in air, as explained in Scheme S2 (SI). In addition, the maximum δ was only 6.1% for the conventional solution-replacement protocol by dissolving the AgNPs with 5 μL of 25% HNO3 and then transferring it into 995 μL of 0.6 M KNO3 (use of 995 μL of 25% HNO3 instead still gave similar results) for preconcentration at −0.3 V for 600 s and LSV stripping. A direct anodic stripping of the cast-coated AgNPs in 0.6 M aqueous KNO3 yielded an experimental δ value of 96.6% (Figure S2, SI). However, at the sandwich-type immunoelectrodes, rather than the bare electrode used here, such a direct ASV protocol D

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change from wine red to purplish red, and the plasmon absorption peak of AuNPs was also weakened and red-shifted, indicating protein-bridged agglomeration of the AuNPs. After adding the silver-staining solution, the suspension showed a gray color and no absorption peak in the visible region but a high absorption peak of HQ at 256 nm (sample 4). After adding 0.2 mM HAuCl4, the suspension showed a light-brown color, because the GRR between the stained silver and HAuCl4 may produce AgCl−Au nanostructures, as shown in eq 2 and discussed below.

(without cathodic preconcentration) must give a signaling efficiency notably lower than our MLAI method, because the electron-insulating immunostructural protein layers between the metal label and the electrode can own a total thickness notably larger than ca. 1 nm (the efficient electron-transfer distance in electrolyte solution as reported before38−40) and the electron communication between the metal label and the electrode can thus be notably blocked (as shown in Scheme S1 and experimentally confirmed in Figure S3, SI). Hence, the in situ metal preconcentration step is very important for improving the signaling efficiency of MLAI. The volume of 25% HNO3 used to dissolve AgNPs was optimized. As shown in Figure S4 (SI), δ decreased with the increase of HNO3 volume, since a smaller volume of HNO3 solution can decrease the diffusion-layer thickness for enhanced preconcentration of atomic silver on the WE and can thus give a larger ASV peak signal. To maximize the signal, we will use 5 μL of HNO3 below. 3.2. Silver Staining and GRRs. In this work, we employed preformed AuNPs to label Ab2 for AuNPs-catalyzed silver staining. The UV−vis spectrophotometric studies on our system are shown in Figure 2. The AuNPs suspension showed

3Ag(s) + AuCl4 −(aq) → Au(s) + x AgCl(s) + (4 − x)Cl−(aq) + (3 − x)Ag +(aq)

(2)

where x must range from 0 to 3. The QCM and CV techniques were used to examine the GRR of interest on the QCM Au electrode. As shown in Figure 3A, we recorded the time course of the QCM frequency during Au-catalyzed silver staining on the QCM Au electrode in stirred ultrapure water under open-circuit condition (note that the Au was sprayed on the unpolished quartz crystal wafer and thus many Au nanostructures existed on the QCM Au electrode surface41). After adding the silver-staining solution, the QCM frequency decreased due to the rapid staining of silver on the bare QCM Au electrode.35 The silver staining was stopped at a final frequency decrease (−Δf 0) of 12.4 kHz here (the mass of stained atomic silver is Δmss = 19.6 μg, as calculated from the Sauerbrey equation).31 After an immediate water rinse and nitrogen drying of the silver-stained QCM Au electrode, a CV experiment as shown in Figure 3B was conducted in 0.6 M aqueous KNO3. On the first positive scan from −0.3 V, an anodic stripping peak of silver was observed at 0.56 V with a peak charge of 18.2 mC (20.4 μg atomic silver, calculated from the Faraday law), and its cathodic peak was at 0.33 V. The mass of silver deposit obtained from the QCM agrees well with that obtained from the silver-stripping peak charge, proving the quantitative veracity of our QCM. Another silver/QCM Au electrode with the same quantity of stained silver was similarly prepared and dried, and we studied the GRR process on its surface in stirred ultrapure water (Figure 3A). After adding the HAuCl4 aqueous solution, the frequency decreased quickly in the first 3 min due to the rapid GRR of stained silver on the Au

Figure 2. UV−vis spectra and digital picture (inset) of the suspensions of 0.50 mL of AuNPs (1), anti-IgG/AuNPs (2), BSA/anti-IgG/AuNPs (3), and the BSA/anti-IgG/AuNPs after being treated by silverstaining solution for 30 min (4) and then HAuCl4 for 10 min (5). Final concentrations: 10 μg mL−1 anti-IgG, 1.0% (w/v) BSA, and 0.2 mM HAuCl4.

a wine red color and an absorption peak at 517 nm (sample 1). After 10 μg mL−1 anti-IgG (sample 2) and then 1.0% BSA (sample 3) were added, the suspension presented a color

Figure 3. (A) Time-dependent QCM frequency responses of a bare QCM Au electrode after adding 1.0 mL silver-staining solution in 1.0 mL of stirred ultrapure water (solid curve) and of a silver/QCM Au electrode after adding 10.0 μL of 50 mM aqueous HAuCl4 in 1.0 mL of stirred ultrapure water (dashed curve). The arrows indicate the moments of adding the silver-staining solution or aqueous HAuCl4. (B) The CV curves before (solid curve, initial potential = −0.3 V, initial scan = positive) and after the GRR (dashed curve, initial potential = 0.2 V, initial scan = positive). Scan rate = 10 mV s−1. E

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Figure 4. SEM images and EDX spectra of antigen/BSA/Ab1/Auplate/GCE (A), Ab2−AuNPs/antigen/BSA/Ab1/Auplate/GCE (B, I), silver/Ab2− AuNPs/antigen/BSA/Ab1/Auplate/GCE (C, J), (Au−silver)1/Ab2−AuNPs/antigen/BSA/Ab1/Auplate/GCE (D, K), silver/(Au−silver)2/Ab2− AuNPs/antigen/BSA/Ab1/Auplate/GCE (E), (Au−silver)3/Ab2−AuNPs/antigen/BSA/Ab1/Auplate/GCE (F), silver/(Au−silver)4/Ab2−AuNPs/ antigen/BSA/Ab1/Auplate/GCE (G, L), and similar four-cycle silver staining−GRR operation plus an additional silver staining on a BSA/Ab1/ Auplate/GCE after its cultivation with Ab2−AuNPs but without preimmobilizing the antigen on BSA/Ab1/Auplate/GCE (H, as an antigen-free control). The 1 μm scale bar in panel E also applies for other SEM images. Concentration of IgG: 4 ng mL−1.

A simple calculation of eq 3 yields x = 2.82 ≈ 3, and thus the final form of eq 2 under our experimental conditions should be

electrode. A final frequency decrease (−Δf 0) of 10.6 kHz (the mass increment is ΔmGRR = 16.8 μg, calculated from the Sauerbrey equation) was obtained after consumption of the stained silver. From the CV experiments shown in Figure 3B, an anodic peak was observed at 0.56 V for oxidation of atomic silver to silver cations on the silver/QCM Au electrode. An additional anodic peak at 0.18 V that should be due to oxidation of atomic silver to AgCl was observed after the GRR on the second anodic scan, and the silver left unreacted during the GRR and the silver obtained from possible AgCl photolysis (the CV experiment was conducted immediately after the GRR) are negligible, as reflected by the lack of an anodic peak at 0.56 V on the first anodic scan. The two anodic peaks at 0.18 V for oxidation of silver to AgCl and at 0.52 V for oxidation of atomic silver to silver cations were also observed in the CV curve with the first negative-going cathodic sweep from the open-circuit potential of 0.2 V, as shown in Figure S5 (SI). Therefore, as shown in Figure 3A, the mass increase of ΔmGRR = 16.8 μg after GRR should correspond to the mass increment due to conversion of stained silver into Au plus AgCl on the electrode, and the mass increase of Δmss = 19.6 μg after silver staining should result from the mass increment due simply to silver staining on the electrode. The combination of the mass increase after silver staining and the mass increase after GRR can be used to make clear the stoichiometry of this GRR, as shown in eq 3 ΔMss ΔMGRR 3 × 107.9 = = −6 Δmss ΔmGRR 19.6 × 10 197 + (107.9 + 35.45)x − 3 × 107.9 = 16.8 × 10−6

3Ag(s) + AuCl4 −(aq) → Au(s) + 3AgCl(s) + Cl−(aq) (4) −

Equation 4 states that the Cl anions have been maximally transformed into AgCl for the GRR on the electrode surface, and this conclusion is thermodynamically supported by the calculation of the precipitation reaction equilibrium based on the solubility product (Ksp) of AgCl, as discussed in the Supporting Information. It should be noted that the formation of AgCl precipitate in this GRR has been mentioned.19,42 As far as we know, however, the concrete reaction equation of this GRR has not been reported before. Here, the combination of QCM and CV techniques as a good quantitative tool has allowed us to delve into the stoichiometry of this GRR on the electrode surface for the first time, which may be extended to stoichiometric studies on other GRRs. Various electrode modifications in our MLAI method were examined in detail. First, taking the AuNPs-labeled immunoassay of IgG as an example, CV, EIS, and QCM were used to investigate various modifications on the WE, as shown in Figures S6 and S7 (SI) with details discussed. These experiments support that all the AuNPs-catalyzed silver-staining reactions and the GRRs on the immunoelectrode were successful. We also used SEM to characterize the fabrication of the immunosensor, and typical results are shown in Figure 4. Here, we take the AuNPs-labeled immunoassay of IgG as an example. We clearly see electrodeposited Au particles on the antigen/ BSA/Ab1/Auplate/GCE (Figure 4A). After the immobilization of Ab2−AuNPs (Figure 4B, Ab2−AuNPs/antigen/BSA/Ab1/ Auplate/GCE), some anchored Ab2−AuNPs granules were observed, proving the successful immunorecognition. After the first AuNPs-catalyzed silver-staining reaction (Figure 4C, silver/Ab2−AuNPs/antigen/BSA/Ab1/Auplate/GCE) and its GRR [Figure 4D, (Au−silver)1/Ab2−AuNPs/antigen/BSA/ Ab1/Auplate/GCE], the Ab2−AuNPs granules become larger and denser. After the third AuNPs-catalyzed silver-staining reaction [Figure 4E, silver/(Au−silver)2/Ab2−AuNPs/antigen/

(3)

where ΔM is the net change of molar mass of the loaded species resulting in the electrode mass change in the reaction (note that the stoichiometric coefficients of eq 2 should be considered), the subscript ss denotes silver staining, 107.9 is the relative atomic mass of silver, 197 is the relative atomic mass of Au, and 35.45 is the relative atomic mass of Cl. F

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the electrode surface to efficiently exchange electrons with the electrode surface, but in our MLAI method, the employed nitric acid can destroy the sandwich immunostructure to allow stained silver and AgCl to touch the electrode surface better to yield electrodeposition of more silver, thus giving the maximum signal, as shown in Schemes 1 and S1 (SI). It is well-known that AgCl is photosensitive and can be photoreduced to atomic silver.43−46 However, little AgCl was degraded to atomic silver after 2 h exposure under the experimental environment, demonstrating that the photolysis of our AgCl to atomic silver was not obvious here (Figure S9, SI). 3.3. Single-Molecule-Level Immunoassay of IgG and AFP. To obtain the best performance of immunoassay, we optimized the number of the silver staining−GRR cycles on the immunoelectrode (Figure 6). Other experimental variables, such as incubation temperature (37 °C here) and pH of incubation solution (PBS, pH 7.4 here), were well-established elsewhere,47 so they were selected according to the references. As shown in Figure 6, the ASV peak current increased obviously with increasing the number of the silver staining− GRR cycles at first and then tended to level off after four silver staining−GRR cycles. This observation can be explained as follows. The GRR can enlarge the sizes of AuNPs to accommodate more atomic silver on the AuNPs surfaces and thus increase the ASV peak current, but an overly large increase in the AuNPs size will suppress its catalytic activity for silver staining on its surface. Hence, four silver staining−GRR cycles plus an additional silver staining were used below. Under the optimum conditions, the ASV peak current is linear with the common logarithm of IgG concentration from 0.4 fg mL−1 to 400 ng mL−1, with a sensitivity of 657 μA dec−1 and a LOD of 0.2 fg mL−1 (five molecules in a 6 μL sample, 6 × 10−6 × 0.2 × 10−15 × 103 × 6.02 × 1023/1.5 × 105 = 4.8 ≈ 5, S/ N = 3) by our MLAI method of four silver staining−GRR cycles plus an additional silver staining, as shown in Figures S10 and S11 (SI) (Figure S11 shows the ASV and calibration curves for low-concentration IgG). If only the gold label/silver staining was used (without GRR), we obtained linearity from 5 fg mL−1 to 500 ng mL−1 with a sensitivity of 100 μA dec−1 and a LOD of 1.2 fg mL−1 (S/N = 3). In contrast, the conventional solution-replacement protocol only gave a linear response from 5 pg mL−1 to 500 ng mL−1 with a sensitivity of 20.1 μA dec−1 and a LOD of 0.1 pg mL−1 (S/N = 3), highlighting the great improvement of analytical performance by our MLAI method versus the existing ones. Our MLAI method has also well balanced the methodological simplicity and sensitivity versus existing methods, as discussed in the SI. In these immunoassays, the responding concentration of the antigen covers several orders of magnitude, so we use a semilogarithmic plot, as reported before.6,48,49 The reproducibility of our MLAI method was evaluated using IgG at three high-concentration levels (0.500, 5.00, and 50.0 ng mL−1) and three lowconcentration levels (0.400, 4.00, and 40.0 fg mL−1), giving relative standard deviations (RSDs) of 6 ± 1% and 13 ± 7%, respectively. The single-molecule-level LOD obtained from our MLAI method here is much better than the literature-reported values for IgG (Table S2, SI) and that experimentally obtained from the conventional solution-replacement protocol (0.1 pg mL−1). To our knowledge, the methods of such a singlemolecule-level LOD or even a single-molecule LOD have been seldom reported for electroanalysis,50 though some singlemolecule-level optical methods have been reported.51−53 We will demonstrate in the next section that it is thermodynami-

BSA/Ab1/Auplate/GCE] and its GRR [Figure 4F, (Au−silver)3/ Ab 2 −AuNPs/antigen/BSA/Ab 1 /Au plate /GCE], the Ab 2 − AuNPs granules become further enlarged and denser. After the four-cycle silver staining−GRR operation plus an additional silver staining, some cubic crystals on the electrode surface were observed [Figure 4G, silver/(Au−silver)4/Ab2−AuNPs/antigen/BSA/Ab1/Auplate/GCE]. As a control, a similar four-cycle silver staining−GRR operation plus an additional silver staining on a BSA/Ab1/Auplate/GCE after its cultivation with Ab2− AuNPs but without preimmobilizing the antigen on BSA/Ab1/ Auplate/GCE (an antigen-free control) hardly changed the electrode surface (Figure 4H), highlighting the importance of AuNPs labeling for the silver staining and GRRs and the negligible effect of Auplate due to the successful blocking of Auplate by the protein layers. The EDX spectra of Ab2−AuNPs/ antigen/BSA/Ab 1 /Au plate/GCE (Figure 4I), silver/Ab2− AuNPs/antigen/BSA/Ab1/Auplate/GCE (Figure 4J), (Au− silver)1/Ab2−AuNPs/antigen/BSA/Ab1/Auplate/GCE (Figure 4K), and silver/(Au−silver)4/Ab2−AuNPs/antigen/BSA/Ab1/ Auplate/GCE (Figure 4L) confirm the increased mass and atomic fractions of silver, Au, and Cl upon increasing the number of silver staining−GRR cycles. Similarly, we used CV to quantify the electroactive AgCl on the immunosensor surface, and typical results are shown in Figure S8 (SI). The cathodic peak current response increased obviously with increasing the number of the silver staining−GRR cycles (first negative-going cathodic sweep from the starting potential of 0.2 V), and the apparent mass of AgCl after each silver staining−GRR cycle can be determined from the Faraday law and the cathodic peak charges for AgCl reduction. The above SEM, EDX and CV characterizations have all proven successful silver-staining reactions and GRRs here. In order to demonstrate the sensitivity-enhancement effect of our MLAI method, some silver/(Au−silver)4/Ab2−AuNPs/ antigen/BSA/Ab1/Auplate/GCEs were prepared for comparison among direct anodic stripping (a), anodic stripping after electroreduction of AgCl under −0.3 V vs SCE for 600 s in 0.6 M KNO3 (b), and our MLAI method (c), as shown in Figure 5. In comparison with our MLAI method, the two protocols of direct anodic stripping and anodic stripping after electroreduction of AgCl give smaller signals. This observation indicates that many of the stained silver and silver chloride have a immunostructure-separated distance too far away from

Figure 5. CV curves and differential pulse ASV curves (inset) at silver/ (Au−silver)4/Ab2−AuNPs/antigen/BSA/Ab1/Auplate/GCE for direct anodic stripping (a), anodic stripping after electroreduction of AgCl under −0.3 V vs SCE for 600 s in 0.6 M KNO3 (b), and our MLAI method (c). Scan rate = 10 mV s−1, initial potential = −0.3 V, initial scan = positive, concentration of IgG: 400 ng mL−1. G

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Figure 6. Differential pulse ASV curves (A) and corresponding curves for optimization the number of the silver staining−GRR cycles on the immunoelectrode (B) (n = 3). Concentration of IgG: 40 ng mL−1.

Figure 7. Differential pulse ASV curves for AFP immunoassay using our GRR-free MLAI method (gold label/silver staining only) (A) or using our MLAI method for four silver staining−GRR cycles plus an additional silver staining (C) and the corresponding calibration curves (B, D) (n = 3).

LOD of 0.1 fg mL−1 (five molecules in a 6 μL sample, 6 × 10−6 × 0.1 × 10−15 × 103 × 6.02 × 1023/6.9 × 104 = 5.2 ≈ 5, S/N = 3). Figure S12 (SI) shows the ASV and calibration curves for low-concentration AFP. The LODs of our method are much better than the experimental result from the conventional solution-replacement protocol (0.4 pg mL−1, Figure S13, SI) and the literature-reported values for AFP (Table S2, SI). Our MLAI method that gives the single-molecule-level LOD of cancer biomarkers has manifested itself as a promising tool for the valuable early detection and treatment of serious diseases, including cancers. We investigated the selectivity of the immunoassay toward AFP (5 fg mL−1) over some possible interfering substances (5 pg mL−1), including IgG, urate oxidase, glucose oxidase, thrombin, and human carcinoembryonic antigen. Remarkably, only AFP induced a significant increase in the electrochemical signal, revealing that the immunoassay is highly selective toward AFP, even at the low concentration, mainly due to the high specificity of anti-human AFP toward AFP (Figure S14, SI). To validate the practicality of the present assay, we also examined

cally feasible to conduct SMLD by the MLAI method, as a theoretical support of our experimental results. Our MLAI method that possesses single-molecule-level LOD of the model analyte IgG is expected to find wide applications in quantitative analysis of samples containing ultratrace analytes. We believe that the single-molecule-level quantitative electroanalysis appears to be an interesting theme worthy of more exploitations in the future. AFP is a very important clinical diagnosis biomarker for liver cancer and rectal cancer and is commonly determined by immunoassay methods.54−56 We used our MLAI method to detect AFP (Figure 7 and S12, SI). Under the optimum conditions, we observed a linear response of ASV peak current to the common logarithm of AFP concentration from 5 fg mL−1 to 500 ng mL−1 with a sensitivity of 99.4 μA dec−1 and a LOD of 1.6 fg mL−1 (S/N = 3) using our GRR-free MLAI method (gold label/silver staining only). In contrast, using our MLAI method for four silver staining−GRR cycles plus an additional silver staining, we obtained linearity from 0.5 fg mL−1 to 500 ng mL−1 with a sensitivity of 675 μA dec−1 and a H

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complex (antigen−Ab1) and a monovalent antibody 2 (Ab2) (i.e., formation of the interfacial sandwich-type Ab2−antigen− Ab1 immunostructure). The initial volume concentrations of interfacial antigen−Ab1, free antigen, and interfacial Ab1 after the above antigen−Ab1 immunological reaction of one single antigen molecule should be 1.1741 × 10−14 M, 0 M, and 1.6612 × 10−3 M (neglect the minor change after formation of one single antigen−Ab1 complex molecule), respectively, and the volume concentration of the dissociated antigen−Ab1 complex at equilibrium is assumed to be y′ M, as shown in eq 7 below

the applicability of our MLAI method to AFP assay in seven clinical human-serum samples (only 6 μL of serum required for each test). Our results agreed well with the hospital results from a chemiluminescence assay (within ±7% RSD), as listed in Table S3 (SI), validating our MLAI method for analysis of target proteins in clinical human sera. We believe that our MLAI method has the promising application potential for analysis of many proteins in extremely small amounts in real samples. 3.4. Discussion on the Thermodynamic Feasibility of Single-Molecule-Level Detection of Proteins by MLAI. As described above, our MLAI method has the experimental ability for SMLD of proteins. Here, we will theoretically demonstrate that our MLAI method for SMLD is thermodynamically feasible. Let us discuss an immunological reaction between one single antigen molecule and an interfacial monovalent Ab1 monolayer at the sandwich-type immunological interface, as shown in eq 5 below

At equilibrium, we obtain eq 8 below K a = 1 × 106 = (1.1741 × 10−14 − y′)/[y′ × (1.6612 × 10−3 + y′)] (8)

A simple calculation of eq 8 yields y′ = 7.0635 × 10−18 M, which is much smaller than the initial concentration of the antigen−Ab1 complex (1.1741 × 10−14 M) and indicates that the antigen−Ab1 complex will almost not dissociate during the washing step or during the following reaction to form the interfacial sandwich-type Ab2−antigen−Ab1 immunostructure. The above calculations regarding equilibrium of the immunological reactions occurring at the electrode|solution interface support that detection of one single antigen molecule on the basis of the interface strategy is thermodynamically possible. However, it should be noted that if the initial concentration of Ab1 in eq 7 is zero (e.g., an ideal homogeneous phase protocol), the antigen−Ab1 complex is dissociated, highlighting that the interface-based quantitative methods, rather than the ideal homogeneous phase ones, have the thermodynamic feasibility for ultrasensitive single-molecule-level bioassay based on equilibrium of the bioaffinity reactions.

where the initial concentration of the interfacial Ab1 monolayer of 1.6612 × 10−3 M is obtained as follows. We assume that Ab1 is of a common protein size of 10 × 10 × 5 nm,57,58 so a full monolayer of Ab1 densely packed on an atomically flat GCE interface of a geometric surface area of 0.0707 cm2 should thus correspond to a monolayer volume of 0.0707 × 10 × 10−7 cm3 = 7.07 × 10−8 cm3 = 7.07 × 10−11 L. The surface coverage of a monolayer Ab1 is equal to 3.3223 × 10−12 mol cm−2 (10 × 5 nm area);57,59 thus, the volume concentration of this densely packed Ab1 monolayer itself should be 3.3223 × 10−12 × 0.0707/(7.07 × 10−11) = 3.3223 × 10−3 M, and the volume concentration of this densely packed Ab1 monolayer allowing the occurrence of its immunological reaction with antigen in an additional monolayer-space (also assuming that the antigen size is d = 10 nm,57,58 as depicted in Scheme S3; Brownianmovement-driven mass transfer) should be 3.3223 × 10−3/2 = 1.6612 × 10−3 M. Obviously, the initial concentration of one immobilized antigen molecule in 7.07 × 10−11 × 2 = 14.14 × 10−11 L solution is 1/(6.02 × 1023 × 14.14 × 10−11) = 1.1748 × 10−14 M. The immunological association constant usually varies from 106 to 1012 M−1 (Ka = 106−1012 M−1).60 Assuming that Ka is equal to 1 × 106 M−1, we can write eq 6 below, by considering that consumption of y M Ab1 and y M antigen can lead to formation of y M antigen−Ab1 complex at the time of antigen− Ab1 immunological reaction equilibrium

4. CONCLUSIONS In summary, we have presented a new MLAI method based on multiple gold label/silver staining and GRRs cycles, followed by simultaneous chemical dissolution/cathodic preconcentration of silver for in situ microliter-droplet ASV detection on the immunoelectrode. Single-molecule-level IgG and AFP can be experimentally detected by our MLAI method. The calculations regarding equilibrium of the immunological reactions occurring at the electrode|solution interface also support that detection of one single antigen molecule based on formation and investigation of the interfacial sandwich immunostructure is thermodynamically possible. Since the in situ ASV detection of one metal nanoparticle after sufficient size enlargement directly on the working electrode can be imagined, the single-moleculelevel detection of a protein by an appropriately designed MLAI method is theoretically and experimentally possible. Immunoassay of AFP in clinical samples by our method gave results agreeable with the hospital results (Table S3, SI). The stoichiometry of the GRR of interest has been discussed in detail by the EQCM technique. Our MLAI method has the advantages of very high sensitivity, very wide linear detection range, single-molecule-level LOD, good accuracy/precision/ stability, easy operation, and small consumption of reagents/ samples, which may have application potential in many fields,

K a = 1 × 106 = y/[(1.1748 × 10−14 − y) × (1.6612 × 10−3 − y)] (6) −14

A simple calculation of eq 6 yields y = 1.1741 × 10 M, which means that the immunological reaction can be carried out completely and that almost all the antigen (here 1.1748 × 10−14 M) can be transformed into the antigen−Ab1 complex (here 1.1741 × 10−14 M) at the interface. Then, let us discuss whether the antigen−Ab1 complex will dissociate or not during the washing step or during the further immunological reaction between the interfacial immunological I

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including clinical assay, food-safety analysis, and environmental analysis.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.5b12004. Supplemental discussion, Schemes S1−S3, Tables S1− S3, and Figures S1−S14 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21475041, 21175042, 21305041 and 21405042), Hunan Lotus Scholars Program (2011), and Hunan Provincial Innovation Foundation For Postgraduate (CX2014B169).

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REFERENCES

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DOI: 10.1021/acs.jpcc.5b12004 J. Phys. Chem. C XXXX, XXX, XXX−XXX